Fig 2: ARG ingestion inhibits ECC-induced proteolysis of Ca2+ regulatory proteins. For the protocols of ARG ingestion and ECC, see legend of Figure 1. (A and E) immunoblot analyses of RyR, DHPR, JP1, and CSQ1 in tibialis anterior muscles. RyR, DHPR, and JP1 were analyzed on the same membrane. For RyR and DHPR, in addition to full-length proteins, faster migrating bands were observed and most likely corresponded to degraded proteins. Total proteins on the membrane, which were used as a loading control, were visualized with Coomassie blue R staining. Positions of molecular mass makers are indicated on the left. (B, C, D, and F) means ± SEM (n = 7 for each muscle) of the contents of full-length RyR (arrow), DHPR, JP1, and CSQ1, respectively. The contents of four proteins investigated were evaluated relative to total proteins. The results are expressed as percentages of the values observed in rested muscles from CON rats. *P < 0.05, versus rested muscles within rats; † P < 0.05, versus ECC muscles from CON rats. ARG, l-arginine; CON, control; CSQ1, calsequestrin-1; DHPR, dihydropyridine receptor; ECC, eccentric contraction; JP1, junctophilin-1; RyR, ryanodine receptor.

Fig 3: TEMR–ADSC construct-repaired LD muscles display structural and functional hallmarks reminiscent of native muscles.Notes: LD muscles repaired with TEMR–ADSC constructs were retrieved 2 months postimplantation and analyzed for morphology and new tissue formation by IHC staining on paraffin-embedded histological sections. IHC staining demonstrated positivity for structural proteins, myosin heavy chain (MF20; A) and titin (9D10; B). Similarly IHC staining revealed the presence of functional proteins, Junctophilin (Jp1; C) and ryanodine receptor 1 (RyR1; D).Abbreviations: TEMR, tissue-engineered muscle repair; ADSC, adipose-derived stem cell; LD, latissimus dorsi; IHC, immunohistochemistry.

Fig 4: Misexpression of triad proteins and expansion of the T-tubule compartment in Ehd1-heterozygous muscle.(A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in Ehd1+/- quadriceps muscle compared to WT correlating with the increase in T-tubule structures. Gel code bands are shown as a loading control (LC). (B) Junctophilin 2 (JP2) protein levels were decreased 13-fold in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (C) Junctophilin 1 (JP1) protein levels were similar in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (D) Ultrastructural analysis reveals ectopic (dotted arrow) and elongated (arrow) T-tubules in 8-week-old Ehd1-heterozygous muscle (Ehd1+/-) stained with potassium ferricyanide to color the T-tubule structures black. (E) Ehd1-heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5µm. (F) Ehd1-heterozygous muscle stained with potassium ferricyanide, outlines duplicated T-tubule structures (two black arrows). Scale 0.5µm. (G) Ultrastructural analysis of 2-D images reveals increased tubule abnormalities in Ehd1-heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).